The present invention relates to a biosensor comprising an electrode and membrane in which the conductance of the membrane changes in response to the presence of an analyte. In particular, the invention relates to the use of composite layer membranes incorporating a supporting layer comprising regions of low ionic or electronic mobility with regions of high ionic or electronic mobility.
International patent application WO 90/08783, the disclosure of which is incorporated herein by reference, discloses a membrane in which the conductance of the membrane is dependent on the presence or absence of an analyte. The membrane comprises a closely packed array of amphiphilic molecules and a plurality of ionophores comprising first and second half membrane spanning monomers. Ionophores within the top layer monomer are capable of lateral diffusion within the membrane. The membrane also includes receptor molecules and the binding of the analyte to the receptor molecules causes a change in the relationship between the first and second half membrane spanning monomers thereby altering the conductivity of the membrane. This type of gating mechanism is referred to as xe2x80x9clateral segregationxe2x80x9d.
In the lateral segregation gating mechanism described in WO 90/08783 it is essential that the membrane include dimeric ion channels with at least one of the monomers being capable of diffusion within the membrane so that the relationship between the two monomers may be altered. The present inventors have developed a biosensor in which gating is achieved by allowing ion channels to diffuse in a lipid membrane between zones of the electrode with differing states of polarisation, conductivity, ion reservoir capacity, or redox potential. On binding of an analyte, the ion channels are locked into one of these regions. With this arrangement any ion channel, membrane spanning or otherwise, can be used.
The provision of the hydrophilic tethers has permitted the construction of a robust membrane but the elimination of that element would further increase the stability of the membrane. This is significant because the membranes themselves are only one or two molecules in thickness. Further, the membranes in use should be able to contact blood and other biological materials that have interfering substances which tend to disturb the integrity of the membrane and its ionic reservoir.
The use of molecular wires in connection with macromolecular devices has been reviewed in J. -M. Lehn, Perspectives in Supramolecular Chemistryxe2x80x94From Molecular Recognition towards Molecular Information Processing and Self-Organisation, (Agnew, Chem. Int. Ed. Engl. 29, 1990, 1304-1319). The molecular wire is a connector permitting electron flow between the different elements of a molecular electronic system. An example is based on caroviologens, long, conjugated, polyolefinic chains bearing pyridinium groups at each end. These have been incorporated into dihexadecyl phosphate vesicles but were not successful as conductors in that environment. Small quantities of zwitterionic caroviologens have been incorporated into phospholipid vesicles and accelerated the rate of reduction of an internal oxidant making it probable that electron conduction occurred. It has also been suggested that one could make polarised molecular wires which would have rectifying properties from conjugated polyolefinic chains, bearing an electron-acceptor group at one end and a donor on the other end.
Highly conducting molecular crystals prepared from porphyrin and phthalocyanine complexes have been prepared as derivatives of the metalloporphyrine skeleton (Hoffman B A and Ibers J A. Porphyrinic Molecular Metals, Acc. Chem. Res. 16, 1983, 15-21).
Molecular wires formed of bispyridium polyenes have been synthesised and incorporated into the bilayer membrane of sodium dihexadecyl phosphate vesicles so that they have formed membrane-spanning electron channels. These molecular wires were not adapted to provide conduction between a membrane and its supporting electrode but had the pyridinium sites close to the negatively charged outer and inner surfaces of the vesicles and the polyene chain crossed the lipidic interior of the membrane. (Arrhenius T S, Blanchard-Desce, M, Dvolaitzky, M, Lehn J-M and Maithete, J. Molecular devices: Caroviologens as an approach to molecular wiresxe2x80x94synthesis and incorporation into vesicle membranes, Proc. Natl Acad. Sci. USA 83, 1986, 5355-5359).
Conducting organic materials similar to and including tetrabenzoporphyrine has been reported in the literature and its electrical properties noted. (Hanack M and Zipplies T. Synthesis and Properties of Doped xcexc Oxo (tetrabenzoporphyrinato) germanium(IV). J. Am. Chem. Soc. 107, 1985, 6127-6129).
In a first aspect the present invention consists in a biosensor comprising an electrode and a membrane, the biosensor including at least two zones each zone differing from each other zone in a property; the membrane including a plurality of ionophores, at least a proportion of the ionophores being capable of lateral diffusion within the membrane, a plurality of first binding partner molecules attached to membrane elements positioned within a first zone such that the first binding partner molecules are prevented from diffusing laterally into a second zone, second binding partner molecules attached to the ionophores, the rate of lateral diffusion within the membrane of the first binding partner molecules and second binding partner molecules being different.
In a preferred embodiment of the invention there is provided an intermediate region between at least portions of the membrane and the electrode, the intermediate region functioning as a reservoir or as a source or sink for ions.
In a further preferred embodiment of the invention the at least two zones of the biosensor are due to differing zones in the electrode, the membrane, the intermediate region or combinations thereof.
In yet another preferred embodiment the property is selected from the group consisting of chemical, polarisation, admittance, ionic reservoir capacity or redox potential.
In a preferred forms of the invention, the electrode comprises a silicon silver composite or a silicon gold composite. Similarly, the electrode may comprise a pattern of silver or gold islands deposited onto silicon oxide or a pattern of silicon oxide islands deposited onto gold or silver. Indeed, a number of possible arrangements will readily occur to those skilled in this area. These include gold/aluminium, silver/gold, silicon rubber/gold, rubber/silver, titanium/gold and niobium/gold, or patterned lipid monolayers attached to the gold so that the lipid regions provide electronic/ionic insulation and the lipid free regions provide an ion reservoir. The essential. criterion is that the electrode comprises zones of differing states of polarisation, conductivity, ionic reservoir capacity or redox potential.
In the situation where the at least two zones of the biosensor comprise a pattern of islands, the islands are preferably arranged to be insulated from each other so that they may be measured independently, or electrically interconnected for simultaneous measurement of all gating sites. In International patent application No PCT/AU89/00352 arrangements for independent measurements are disclosed. This disclosure of PCT/AU89/00352 is hereby incorporated by reference.
In yet a further preferred embodiment of the present invention, the first and second zones comprise two interleaved but separated comb electrodes at different potentials. Preferably, the separation between adjacent teeth of the respective combs and the width of each tooth is less than one micron. The total number of teeth on each electrode is preferably approximately 500.
The first and second binding partner molecules may be attached to the membrane elements and ionophores by any of the techniques disclosed in Australian patent No 617687 or PCT/AU93/00509, the disclosures of which are incorporated by reference. The first binding molecules are attached to membrane elements which span the membrane. Preferred membrane elements are bolar lipids or membrane spanning proteins.
The first binding molecule is prevented from diffusing laterally within the membrane by attachment of the membrane element which is attached to the electrode. For example, in the preferred forms of the invention, the membrane element is an bolar lipid or membrane spanning protein which is attached to the electrode.
The membrane elements to which the first binding partner moieties are attached may be differentially attached to preselected zones of the electrode by selection of the attachment group. For example where the electrode is a gold/silicon and it is desired to position the first binding moieties over the gold zones of the electrode, the membrane element is provided with a thiol or disulphide group such that membrane element is attached to gold zones of the electrode by chemisorption.
Alternatively, the attachment group may be selected such that the membrane element is attached to the silicon zones.
The differing zones may also be created by taking advantage of the self-assembly properties of the molecules themselves. The adsorption of molecules onto substrates is dictated by the type of functional group involved in the substrate-molecule bond, the overall structure of the molecule, the size of the molecule, and the ratio of molecules when mixtures are used, all of which affect the kinetics of adsorption for each type of molecule and can influence the formation of phase-separated molecular domains or zones. PCT/AU93/00509 xe2x80x9cImproved Sensor Membranesxe2x80x9d and PCT/AU96/00369 xe2x80x9cSelf-assembly of sensor membranesxe2x80x9d (the disclosures of which are incorporated herein by reference) show that the function of the biosensor membrane can be dependent on the ratio of tethered lipid to small spacer molecule which adsorbs to the gold substrate. For example, the optimum ratio for DLP (Linker Lipid A) and MAAD (mercaptoacetic acid) in the biosensor membrane is 2:1. If the MAAD concentration is increased by a factor of two or more, then the percentage of bilayer membrane which forms a xe2x80x98floatingxe2x80x99 bilayer or non-tethered zone due to the greater amount of MAAD) molecules adsorbed onto the gold surface, increases, which in turn decreases the stability and specific response of the biosensor to analyte detection, and can affect the reservoir properties within the tethered and non-tethered regions.
Separate zones can also be formed by using mixed substrates (e.g. selectively etched metal alloys or layered metals, selective substrates prepared by conventional lithography methods, etc), and attaching substrate-specific functional groups onto molecules which are be tethered to the substrate in specific zones.
Selecting different types of molecules with chemical groups or bonds which can be selectively broken by application of energy in the form of heat, light, UV, laser, etc., after deposition onto a uniform substrate, can also result in the formation of different zones, the size of which can be controlled by the ratio of the different molecules.
It is also possible to form the differing zones using micro-patterning of self assembled monolayers (SAMs) on a solid substrate. Details regarding this procedure can be found in xe2x80x9cSelf-Organization of Organic Liquids on Patterned Self-Assembled Monolayerdxe2x80x9d by Hans A. Biebuyck and George M. Whitesides; Langmuir 1994, 10, 2790-2793 and xe2x80x9cScanning Force Microscopies Can Image patterned Self-Assembled Monolayersxe2x80x9d by James L. Wilbur, Hans A. Biebuyck, John C. MacDonald and George M. Whitesides; Langmuir 1995, 11, 825-831 (the disclosures of these articles are included herein by reference).
Briefly, the patterning is done using a silicone rubber stamp with which a SAMs can be transferred to specific areas of a substrate. The uncoated areas can then be filled in with another SAM. When the two SAMs have different endgroups one can use lateral force microscopy to distinguish the regions of each SAM. An analogous technique could be used to form the two regions (conducting and non-conducting) of the composite substrate.
In yet another preferred embodiment of the present invention there is provided an intermediate region between at least portions of the membrane and the electrode, the intermediate region functioning as a reservoir or as a source or sink for ions. As will be readily apparent to those skilled in the art the provision of such an intermediate region between selected regions of the membrane and electrode will result in regions or zones of differing reservoir capacity. Such an arrangement may be achieved by using an electrode having xe2x80x9cpeaksxe2x80x9d and xe2x80x9ctroughsxe2x80x9d in which the membrane extends from xe2x80x9cpeakxe2x80x9d to xe2x80x9cpeakxe2x80x9d with an intermediate region being provide in each of the intervening xe2x80x9ctroughsxe2x80x9d.
The intermediate region may an ionic reservoir such as are disclosed in International Patent Application Nos. PCT/AU92/00132, PCT/AU93/00509 and PCT/AU96/00304. (The disclosures of these applications are incorporated herein by reference.).
In an alternative embodiment the intermediate region comprises molecular wires.
The molecular wires preferably have a porphyrinic or octathiophene based structure. The porphyrinic structure, which is preferred, is preferably comprised of a series of fused porphyrin rings with adjacent components of the wire fused through the [b] bond of the porphyrinic ring. This type of orientation of connectivity results in the movement of the electrons through the large molecular orbitals of the molecular wire rather than via the xcfx80-xcfx80 interactions seen in other macrocyclic conductors. Each porphyrin ring in the molecular wire is preferably substituted in its four meso positions. The substituents at the four meso positions preferably stabilise the porphyrin ring, solubilise the porphyrin and/or subsequent derivatives and provide an electrically insulating sheath around the core of the molecular wire. Preferably, the substituent at each of the four meso positions is a 3,5-di-tert-butylphenyl ring.
The molecular wire further preferably includes bridging units between two adjacent porphyrin rings and between a porphyrin ring and end functional groups. The bridging units are preferably substantially rigid such that the molecular wire cannot fold back on itself. The bridging units also preferably provide a conjugated and planar pathway for electrons between adjacent components of the molecular wire. Preferred examples of the bridging units include an anthracene unit or tetraazaanthracene unit fused at the [b] and [e] bonds of the parent tetraazaanthracene system to the other components on the wire.
The porphyrin rings in the molecular wire may exist as freebase or as the metal chelate. In any particular molecular wire, any combination of metalloporphyrinic or freebase porphyrinic rings may be used. Metal chelation of the porphyrin rings in a molecular wire allows finer modulation of the electronic properties of the wire. A preferred chelate metal is copper.
Preferably, the molecular wires are tethered to the surface of the electrode through a binding group. Where the electrode includes gold regions this may be achieved using binding groups having a nitrogen or sulphur bearing compound. Preferred examples of such compounds include a [d] fused 1,10-phenanthroline group, a benzimidazolopyridyl group, a phenyl substituted benzimidazolophenyl group, a bisethylthio phenyl group or multiples and combinations thereof.
In a preferred embodiment of this invention, the intermediate region comprising molecular wires (MW) are formed by self-assembly onto the electrode surface. This renders the surface very hydrophobic and facilitates the subsequent formation of a lipid monolayer or lipid bilayer membrane having ionophores whose conductivity to ions responds differentially to the presence of an analyte.
As with the ionic reservoir where the intermediate region comprises molecular wires it is possible to form the differing zones by manipulation of regions of the molecular wire layer. For example, one could incorporate photosensitive moieties into a molecular wire so that exposure to some wavelength of light could disrupt the conducting pathway. Molecular wires with a quinone in the molecular backbone which are expected to undergo a (reversible) isomerisation when exposed to light that should effect the electronic flow could be used. In a similar manner it could be arranged (by suitable synthesis) that the molecular changes are permanent after illumination so that one can use a xe2x80x9cphoto-lithographicxe2x80x9d type of exposure with a mask dictating xe2x80x9cpatterningxe2x80x9d of the molecular conductivity across the substrate surface.
The molecular wire layer functions as a reservoir or as a source or sink for ions, with the following potential advantages:
(1) A tether region comprising a hydrophilic ionic reservoir is not required between the conductive gold surface and the lipid membrane;
(2) The membrane is rendered more stable, offering storage over long periods without degradation;
(3) Greater capacity for ionic storage than is achieved by creation of aqueous compartments;
(4) Facilitates the assembly of lipid monolayer or bilayer membranes.
A conducting bilayer (molecular wire plus lipid) is produced when a combination of conducting molecular wire and ionophore is made. The molecular wire containing monolayer, therefore acts as a robust and solid ion complexing layer which can effectively replace the tethered ion reservoir. Alternatively, the molecular wire may increase the effectiveness of the conductor/ion interface by virtue or high electronic conduction along the molecular wires.
As used herein the term xe2x80x9cbinding partner moleculexe2x80x9d is used in its widest context. The binding partner molecule may be any chemical entity capable of binding to the desired analyte. It is any compound or composition capable of recognising another molecule and represents half of a binding pair.
Binding pairs include antibodies and either an anti-antibody, the antigen recognised by that particular antibody or an analyte analogue; naturally occurring soluble or cellular receptors and the molecules recognised by them; enzymes and their substrates, inhibitors or analogues of these; lectins and carbohydrates, chelating agents and ions, for example, calcium would be the partner to the chelating agent EDTA.
In a preferred embodiment of the present invention the first and second binding pair molecules are antibodies or any active binding fragments of antibodies.
In further preferred embodiments of the present invention the ionophore is gramicidin or analogues thereof or valinomycin.
The membrane may be single or multi-layer, however, it is presently preferred that the membrane is either a monolayer or bilayer. Where the membrane is a bilayer the ionophore may be a gramicidin dimer.
In yet another preferred form of the invention the membrane elements extend through the membrane. It is also preferred that the membrane elements are attached to the electrode via attachment groups.
The diffusion of the gramicidin ion channels in the membrane can be free or forced by external influences such as magnetic or electrostatic fields. Magnetic particles bound to an antibody may be used to link an analyte to the ion channel which could then be moved between different regions of the electrode to provide electrical gating or signal modification. Electrophoretic methods may be used in a similar manner.
In a second aspect, the present invention consists in a method of assaying a sample for the presence of an analyte, the method comprising contacting the biosensor of the first aspect of the present invention in which the first and second binding partner molecules bind to the analyte with the sample and measuring the conductivity of the membrane.
In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the accompanying Examples and Figures.